Like the case found in Europe and the United States, the number of colon cancer patients in Japan is rapidly increasing from year to year, and the colon cancer is now accounting for the major parts of the mortality caused by cancer. It is thought that this is due to the change in the eating habits of Japanese people to eat more meat, just like the people in the Western world. More specifically, about 60 thousand people are developing colon cancer every year, and also in terms of the number of deaths caused by cancer in different organs, colon cancer comes in 3rd following gastric cancer and lung cancer, and this number is expected to increase even further in the future. On the other hand, unlike other cancers, nearly 100% of colon cancer can be cured if treated at an early stage of development. Therefore, it is extremely worthwhile to select colon cancer as a subject for the screening of early stage cancers, and studies for developing examination methods for the early detection of colon cancers have been intensively conducted.
As the examination methods for the early detection of colon cancers, for example, enema examinations, colonoscopic examinations and the like have been carried out. An enema examination refers to an examination process in which barium is injected into the large intestine and caused to attach to its mucosal surface, and X-ray is irradiated thereto to perform radiography of the surface irregularities, thereby observing the surface of large intestine. On the other hand, a colonoscopic examination refers to an examination process in which the inside of a large intestine is directly observed using an endoscope. The colonoscopic examination in particular is highly sensitive and specific, and is also advantageous in that the removal of polyps or early stage cancers is possible.
However, these test methods are costly and impose a heavy burden on the subjects, and they are also associated with the risk of complications. For example, the enema examination is associated with the risks of X-ray exposure and intestinal obstruction. In addition, the colonoscopic examination is an invasive process since an endoscope is directly inserted inside the large intestine, and the endoscopic operations also require highly technical skills, and thus facilities that can provide this type of examination are limited in number. For this reason, these examination methods are not suitable for colon cancer screening of members of the general public who have no symptoms in cases such as routine checkups.
In recent years, as a primary screening method for colon cancer, a fecal occult blood test has been widely performed, which is a non-invasive process and can be performed at a low cost. The fecal occult blood test is a method for examining the presence and absence of the hemoglobin contained in stool and originating from red blood cells, thereby indirectly predicting the presence of colon cancer. The fecal occult blood test has been widely used for the following reasons: i.e., the collection and storage of stool can be conducted at normal temperatures; no special storage conditions such as refrigeration and freezing are required; and the test can be easily carried out in ordinary households and the operation therefor is highly simple and easy. However, the sensitivity of the fecal occult blood test is low at about 25%, which means that there is a relatively high risk of overlooking the presence of colon cancers. In addition, its positive predictive value is also low, and the percentage of actual colon cancer patients among the subjects that are determined to be positive in a fecal occult blood test is 10% or less. Accordingly, the test is fraught with many false positives. For this reason, there is a strong demand for the development of novel examination methods that are more reliable.
As a new examination method suited for routine checkups or the like which is noninvasive, simple and easy, and also more reliable, a test for examining the presence and absence of cancer cells or genes originated from cancer cells in stool is gaining attention. As compared to the fecal occult blood test, whereby the presence of bleeding from the gastrointestinal tract which is caused indirectly in association with the development of colon cancer is examined, this method is expected to constitute a more reliable examination method because it involves a direct examination concerning the presence and absence of cancer cells or genes originated from cancer cells.
In order to detect cancer cells or the like in stool samples with high accuracy, it is important to efficiently recover the nucleic acid originating from the cancer cells in the stool samples. The amount of nucleic acids originating from cancer cells is particularly low, and nucleic acids are also extremely prone to degradation since a large amount of digestion residues or bacterial cells are contained in stool. Therefore, in order to efficiently recover the nucleic acid, especially the nucleic acid originating from mammalian cells such as human cells, from the stool samples, it is important to prepare the stool samples so as to prevent the degradation of nucleic acids in the stool and to stably preserve the samples until the time of examination operations. As such methods for preparing stool samples, for example, there is a method in which the cancer cells exfoliated from the gastrointestinal tracts such as large intestines are isolated from the collected stool. By isolating cancer cells from stool, the adverse effects caused by the degrading enzymes originating from bacteria and the like such as proteases, DNases and RNases can be suppressed. As a method to isolate cancer cells from stool, for example, a method has been disclosed, which is a method for isolating cells and characterized by including: a) a step for cooling stool down to a temperature below its gel freezing point; and b) a step for collecting cells from the stool while maintaining the stool at a temperature below its gel freezing point, such that the stool remains substantially intact (for example, refer to Patent Document 1). As an alternative, a method has been disclosed in which stool is dispersed in a transport medium containing a protease inhibiting substance, a mucilage solubilizing agent and a bactericide at a normal ambient temperature, followed by the isolation of cells exfoliated from the large intestine (for example, refer to Patent Document 2).
On the other hand, when observing cell forms histologically and cytologically, in order to maintain the form of collected cells until the time of observation, various fixation methods such as formalin fixation and alcohol fixation have conventionally been performed. As a preservation solution that enables the long term preservation of mammalian cell samples as well as the cell observation following the preservation which takes advantage of the above fixation methods, for example, a cell solution preserving agent has been disclosed which contains an alcohol miscible with water in an amount sufficient for fixing mammalian cells, an anticoagulant in an amount sufficient for preventing the aggregation of mammalian cells in the solution, and a buffering agent that keeps the solution pH within a range from 4 to 7 (for example, refer to Patent Document 3).
Further, as a preserving solution that enables not only the histological and cytological observations of cells, but also the molecular biological analysis of proteins, nucleic acids and the like in the cells following preservation, for example, a universal collection medium containing a buffer component, at least one alcohol component, a fixative component, and a chemical agent which suppresses the degradation of at least one type of molecules selected from the group consisting of RNA, DNA and proteins (for example, refer to Patent Document 4), a non-aqueous solution containing 5 to 20% of polyethylene glycol and 80 to 95% of methanol (for example, refer to Patent Document 5), or the like has been disclosed. In addition, a composition is also disclosed which is a composition that stabilizes the cell structure and nucleic acids and contains: (a) a first substance that contains at least one type of alcohol or ketone and is capable of precipitating or denaturing proteins; and (b) a second promoting substance which promotes the infusion of the first substance into at least one cell (for example, refer to Patent Document 6).
[Patent Document 1] Published Japanese Translation No. Hei 11-511982 of PCT International Publication
[Patent Document 2] Published Japanese Translation No. 2004-519202 of PCT International Publication
[Patent Document 3] Japanese Unexamined Patent Application, First Publication No. 2003-153688
[Patent Document 4] Published Japanese Translation No. 2004-500897 of PCT International Publication
[Patent Document 5] Published Japanese Translation No. 2005-532824 of PCT International Publication
[Patent Document 6] Japanese Unexamined Patent Application, First Publication No. 2001-128662
[Patent Document 7] Japanese Patent Application, Second Publication No. Hei 6-72837